(175as) Polyanhydride Nanoparticle Encapsulation Preserves Stability and Antigenicity of Mucin-Based Antigen upon Release

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasingly challenging threat, especially with an aging population. Unlike many other cancers, conventional chemotherapy has been ineffective in treating pancreatic cancer. Because of the early metastatic nature and resistance to traditional treatments, effective therapies to eradicate tumorigenesis are essential. In this regard, immunotherapies have received significant attention wherein the identification and establishment of potent pancreatic neoantigens or upregulated tumor antigens, such as mucins, is critical. During PC progression, MUC4 and MUC16 are the most differentially expressed membrane-bound mucins compared to normal pancreas and pancreatitis and are aberrantly glycosylated. In the current study, the differentially expressed transmembrane tumor antigen MUC4β was encapsulated in polyanhydride nanoparticles and evaluated for its stability during the synthesis and its antigenicity, upon release over the course of 30 days. The general premise is that the tumor-associated antigen (TAA) will be delivered via the nanoparticle carrier, captured by antigen presenting cells, and presented to immature T cells in order to induce TAA-specific cytotoxicity T cells. Often times, TAA-specific antibody may contribute to the immunotherapy via antibody-dependent cytotoxicity (ADCC) and be activated by activation of complement mediated killing. In this regard, MUC4β stability and antigenicity become vital for successful induction of immunotherapy. In our work, polyanhydride nanoparticles were used as an antigen carrier platform due to their superior surface erodible mechanism, tunable amphiphilic chemistry, less acidic degradation products, and high cellular internalization. Amphiphilic polyanhydride copolymers based on 20 mol% 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 80 mol% 1,6-bis(p-carboxyphenoxy)hexane (CPH), were used to assess MUC4β stability and release kinetics as well as to demonstrate activation of antigen presenting cells (e.g., dendritic cells). MUC4β was cloned, expressed in E.coli and purified. The protein was shown to be released in a sustained manner from the nanoparticles. A burst release was characterized in the first 3 days along with a near first order release afterwards. The total encapsulation efficiency was determined to be 41%. MUC4β’s stability and antigenicity were characterized by SDS-PAGE, fluorescence spectroscopy, and ELISA. Over 90% preservation of tertiary structure and antigenicity was verified. Further, mice were immunized with multiple formulations of MUC4β encapsulated nanoparticles in order to evaluate the MUC4β-specific antibody responses. The data demonstrated that the nanoparticles successfully preserved the structure and antigenicity of the released MUC4β, which also resulted in the induction of serum antibody responses. Altogether, these results lay the foundation for future evaluation of this platform for pancreatic cancer immunotherapy.